scholarly journals Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

2021 ◽  
Vol 17 (1) ◽  
pp. e1009227
Author(s):  
Dani A. C. Heesterbeek ◽  
Remy M. Muts ◽  
Vincent P. van Hensbergen ◽  
Pieter de Saint Aulaire ◽  
Tom Wennekes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Immune System ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Human Neutrophils ◽  
Gram Negative Bacteria ◽  
Antimicrobial Proteins ◽  
Gram Negative

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

scholarly journals Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

2020 ◽  
Author(s):  
Dani A. C. Heesterbeek ◽  
Remy M. Muts ◽  
Vincent P. van Hensbergen ◽  
Pieter de Saint Aulaire ◽  
Tom Wennekes ◽  
...  
Keyword(s):  
Immune System ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Damage ◽  
Gram Negative Bacteria ◽  
Human Immune System ◽  
Antimicrobial Proteins ◽  
Gram Negative ◽  
E Coli ◽  
Human Immune

AbstractInfections with Gram-negative bacteria form an increasing risk for human health, which is mainly due to the increase in antibiotic resistance. The cell envelope of Gram-negative bacteria consists of an inner membrane, a peptidoglycan layer and an outer membrane, which forms an impermeable barrier to many antibiotics and antimicrobial proteins. The complement system is an important factor of the human immune system that can efficiently kill Gram-negative bacteria via the formation of large pores in the bacterial outer membrane, called Membrane Attack Complexes (MACs). To better understand how these MAC pores damage the complex cell envelope of Gram-negative bacteria, we recently developed a fluorescent reporter system to study membrane damage in E. coli. Here, we used a similar experimental setup in combination with flow cytometry, confocal microscopy and conventional plating assays to elucidate how different components of the immune system act synergistically to effectively clear invading bacteria. We demonstrate how MAC-dependent outer membrane damage enhances the susceptibility of E. coli to further degradation of the cell envelope by lysozyme, leading to drastic changes in the morphology of these bacteria. Furthermore, we elucidate that the MAC enhances the susceptibility of E. coli to phospholipases, and to degradation and killing inside human neutrophils. Altogether, this study provides a detailed overview on how different players of the human immune system work closely together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate, or work synergistically with the immune system.

Infectious disease

2019 ◽  
Author(s):  
Stevan R. Emmett ◽  
Nicola Hill ◽  
Federico Dajas-Bailador
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Nucleic Acid ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Antibiotics include an extensive range of agents able to kill or prevent reproduction of bacteria in the body, without being overly toxic to the patient. Traditionally derived from living organisms, most are now chemically synthesized and act to disrupt the integrity of the bacterial cell wall, or penetrate the cell and disrupt protein synthesis or nucleic acid replication. Typically, bacteria are identified according to their ap­pearance under the microscope depending on shape and response to the Gram stain test. Further identification is obtained by growth characteristics on various types of culture media, based on broth or agar, biochemical and immunological profiles. Further testing on broth or agar determines antibiotic sensitivity to guide on anti­biotic therapy in individual patients. This process can take 24– 48 hours to culture and a further 24– 48 hours to measure sensitivities. Increasingly, new technology, e.g. Matrix Assisted Laser Desorption Ionization— Time of Flight (MALDI- TOF) and nucleic acid amplification as­says, are being used to provide more rapid identification. The Gram classification, however, is still widely referred to as it differentiates bacteria by the presence or absence of the outer lipid membrane (see Figure 11.1), a fundamental characteristic that influences antibiotic management. Antimicrobial agents rely on selective action exploiting genetic differences between bacterial and eukaryotic cells. They target bacterial cell wall synthesis, bacterial protein synthesis, microbial DNA or RNA synthesis, by acting on bacterial cell metabolic pathways or by inhibiting the ac­tion of a bacterial toxin (see Table 11.1). Both Gram- positive and Gram- negative bacteria possess a rigid cell wall able to protect the bacteria from varying osmotic pressures (Figure 11.1). Peptidoglycan gives the cell wall its rigidity and is composed of a glycan chain of complex alternating carbohydrates, N- acetylglucosamide (N- ATG), and N- acetylmurcarinic acid (N- ATM), that are cross- linked by peptide (or glycine) chains. In Gram-positive bacteria, the cell wall contains multiple peptido­glycan layers, interspersed with teichoic acids, whereas Gram- negative bacteria contain only one or two peptido­glycan layers that are surrounded by an outer membrane attached by lipoproteins. The outer membrane contains porins (which regulate transport of substances into and out of the cell), lipopolysaccharides, and outer proteins in a phospholipid bilayer. For both Gram- negative and Gram-positive bacteria, peptidoglycan synthesis involves about 30 bacterial enzymes acting over three stages. Since the cell wall is unique to bacteria, it makes a suitable target for antibiotic therapy.

scholarly journals Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

2021 ◽  
Vol 17 (11) ◽  
pp. e1010051
Author(s):  
Dennis J. Doorduijn ◽  
Dani A. C. Heesterbeek ◽  
Maartje Ruyken ◽  
Carla J. C. de Haas ◽  
Daphne A. C. Stapels ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Complement Proteins ◽  
Bacterial Cell Envelope ◽  
Complement Resistance

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

scholarly journals Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

2021 ◽  
Author(s):  
Dennis J. Doorduijn ◽  
Dani A.C. Heesterbeek ◽  
Maartje Ruyken ◽  
Carla J.C. de Haas ◽  
Daphne A.C. Stapels ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Complement Proteins ◽  
Bacterial Cell Envelope

Complement proteins can form Membrane Attack Complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria, because a robust way to specifically prevent polymerization of C9 has been lacking. In this paper, polymerization of C9 was prevented without affecting the binding of C9 to C5b-8 by locking the first transmembrane helix domain of C9. We show that polymerization of C9 strongly enhanced bacterial cell envelope damage and killing by MAC pores for several Escherichia coli and Klebsiella strains. Moreover, we show that polymerization of C9 is impaired on complement-resistant E. coli strains that survive killing by MAC pores. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

scholarly journals Peptidoglycan Remodeling EnablesEscherichiacoliTo Survive Severe Outer Membrane Assembly Defect

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Niccolò Morè ◽  
Alessandra M. Martorana ◽  
Jacob Biboy ◽  
Christian Otten ◽  
Matthias Winkle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Cross Links ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show thatEscherichia colicells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCEIn Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show thatEscherichia colicells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

scholarly journals How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

BioEssays ◽  
2019 ◽  
Vol 41 (10) ◽  
pp. 1900074 ◽  
Author(s):  
Dennis J. Doorduijn ◽  
Suzan H. M. Rooijakkers ◽  
Dani A. C. Heesterbeek
Keyword(s):  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bacterial Cell Envelope

scholarly journals The ColM Family, Polymorphic Toxins Breaching the Bacterial Cell Wall

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten G. K. Ghequire ◽  
Susan K. Buchanan ◽  
René De Mot
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cytoplasmic Membrane ◽  
Catalytic Domain ◽  
Gram Negative Bacteria ◽  
Antibacterial Peptides ◽  
Gram Negative ◽  
Lipid Ii ◽  
Associated Proteins ◽  
Colicin M

ABSTRACT Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM) targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities.

scholarly journals The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

2008 ◽  
Vol 190 (6) ◽  
pp. 2065-2074 ◽  
Author(s):  
Mary E. Laubacher ◽  
Sarah E. Ades
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Stress Responses ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Envelope Stress ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

scholarly journals Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis

mBio ◽  
2021 ◽  
Author(s):  
Brent W. Simpson ◽  
Marta Nieckarz ◽  
Victor Pinedo ◽  
Amanda B. McLean ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Mechanical Strength ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Structural Support ◽  
Peptidoglycan Synthesis

Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

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